Project Summary/Abstract The emergence of mosquito-borne viruses is, unfortunately, not rare in modern society. This is exemplified by the introduction and rapid spread of West Nile virus in North America during the late 1990's, and chikungunya virus in the Indian Ocean region and the Americas. The primary facilitators of emergence are human activities, including globalization, climate change, and urbanization. In the examples of West Nile and chikungunya viruses, however, successful establishment was partially attributed to the virus adapting to local environments. Now, Zika virus has emerged within the Americas and threatens to infect millions. Compounding the situation, is the recently discovered association of the virus with severe congenital defects and neuropathologies. The unexpected scale and severity of the Zika virus epidemic led several scientists to hypothesize that this may also be related to recent evolutionary events in the virus. The keys to understanding functional virus evolution during epidemics are (1) genomic surveillance and (2) experimental investigation of functional aspects of virus genetics. The lack of Zika virus genetic data, however, means that evolutionary hypotheses of Zika virus emergence remain untested. Via large-scale collaborations, we recently developed methods to successfully sequence Zika virus directly from clinical and mosquito samples, which led to a major expansion of available genomic data of the virus. As part of this work, we identified several Zika virus amino acid changes that dominate in distinct geographical regions. In this proposal, we will assess functional and fitness effects related to Zika virus mutations in mosquito and human environments. Combined with monitoring their spread via continued genomic surveillance, we will develop a model to investigate if the Zika virus epidemic could have been augmented by functional virus evolution. We propose the following aims: Aim 1: In-depth genomic surveillance of the Zika virus epidemic. By sequencing Zika virus from mosquito and clinical samples, we will (1) continuously monitor virus evolution, (2) create a large catalog of virus mutations, (3) identify where each mutation is geographically present, and (4) evaluate the epidemic process. Aim 2: Fitness of Zika virus mutations circulating in the Americas. Using a reverse genetics platform, we will experimentally evaluate the fitness and function of each identified clade-defining mutation in mosquito and human experimental systems, to determine how they impact transmission and clinical disease. Our proposed aims will provide insights into the genetic determinants of Zika virus disease and emergence. Moreover, the studies outlined here will provide a framework for evaluating functional virus evolution during fast-paced outbreaks.